Neuropeptides are involved in many biological processes, including reward mechanisms and memory. Several peptides are thought to contribute to the physiological response to some drugs of abuse, and it is likely that additional peptides are involved. The overall focus of this grant application is to study neuropeptides and the processing enzymes that produce the neuropeptides, with an emphasis on the effects of drugs of abuse on peptide levels and processing. Peptide levels are typically measured by radioimmuno or radioreceptor assays. While sensitive, these techniques have several restrictions;the most serious is that they are limited to known peptides and a specific question. Recent developments in mass spectrometry and isotopic labeling have led to a fundamental change in the ways that peptides can be measured. Now, it is possible to know the precise molecular form of the peptide being measured. Furthermore/the approach can detect a wide range of peptides, including novel ones. Using a quantitative mass spectrometry-based peptidomics technique, over 100 peptides can be detected in a single experiment. This technique will be used in Aim 1 to evaluate the physiological role of various peptide processing enzymes by examining relative levels of peptides in mutant or 'knock-out'mice that lack carboxypeptidase E or prohormone convertase activity. The peptidomics technique will be used in Aim 2 to determine which peptides are up- or down-regulated by chronic and acute treatment of wild type mice with cocaine, morphine, or methamphetamine. Aim 3 will investigate the physiological role of peptides found in Aims 1 and 2, with a focus on paradigms related to drug abuse. Collectively, these studies will greatly expand our knowledge about neuropeptides and neuropeptide processing enzymes in general, and will identify targets for many further studies on the role of particular peptides in the short- and long-term adaptations to drugs of abuse.